1. Field of the Invention
This invention relates to living cells encapsulated in a polymeric membrane. More particularly, this invention relates to living cells encapsulated in a cohesive permselective membrane comprising a biopolymer and a polyelectrolyte.
2. Description of Related Art
The use of microencapsulated cells as hybrid artificial organs was first proposed in 1964. Endocrine cells, islets, and hepatocytes were proposed to be encapsulated by micropheres formed by the complexation between alginate and calcium. [Chang, T. M. S., Artificial Cells, 1972, Springfield, Ill. Charles C. Thomas.] Intensive studies of these artificial cells, however, began only in the last decade; earlier studies failed to produce semipermeable microcapsules that have the right permeability and soft tissue biocompatibility. In the 1980's, islets of Langerhans were encapsulated in alginate-poly-l-lysine-alginate capsules. [Lim, F. and A. M. Sun, Microencapsulated islets as bioartificial endocrine pancreas. Science, 1980. 210: p. 908.] By using purer alginate and more viscous alginate solutions, researcher obtained microcapsules that were impermeable to normal serum immunoglobulin. [Goosen, M. F. A., et al., Optimization of microencapsulation parameters: Semipermeable microcapsules as a bioartificial pancreas. Biotech. Bioeng., 1985.27: p. 146.] Implantation in the intraperitoneal space of diabetic rats also reversed the diabetic state of some animals for up to one year.
Continued refinement of the system led to restoration of normoglycemia by microencapsulated porcine islets in 13 to 18 mice for up to ten months [Sun, A. M., et al., In vitro and in vivo evaluation of microencapsulated porcine islets. ASAIO J, 1992. 38: p. 125.] Individually encapsulated rat islets placed in the intercapillary space of hollow fibers made of poly(acrylonitrile-co-vinyl chloride) were implanted into diabetic rats. [Lacy, P. E., et al., Maintenance of normoglycemia in diabetic mice by subcutaneous xenografts of encapsulated islets. Science, 1991. 254: p. 1782.] Over 80 percent of the animals maintained normoglycemia for at least 60 days. These systems remain promising as a clinical treatment for diabetes mellitus. [Calafiore, R., Transplantation of microencapsulated pancreatic human islets for therapy of diabetes mellitus. ASAIO J, 1992.38: p. 34.]
Another promising use of these microencapsulation systems is in construction of hybrid artificial liver. Current therapy for fulminant hepatic failure is plagued by severe donor shortage and complications associated with liver transplantation. Hepatocyte transplantation represents an attractive alternative. The major hurdle is immunological rejection, which potentially can be resolved by microencapsulation. Even in a hollow fiber configuration, microencapsulation might still be necessary and advantageous over free hepatocytes because aggregated hepatocytes might remain viable and functional for a longer period of time.
Rat hepatocytes encapsulated by the above-described alginate-polylysine system has been shown in vitro to maintain part of the functions for up to five weeks. [Cai, Z., et al., Development and evaluation of a system of microencapsulation of primary rat hepatocytes. Hepatology, 1989. 10: p. 855.] In this case, however, the nature of the substrate may be more crucial than the case for artificial pancreas. Hepatocyte does not proliferate in culture and loses many of its differentiated functions rapidly. The alginate-polylysine substrate appears to be inferior to collagen-coated substrate in maintaining those term functions, and that remains to be the greatest challenge.
Pertinent to the goals of rehabilitation engineering is implantation of polymer encapsulated neurotransmitter secreting cells for various central nervous system (CNS) deficits. This is an attractive idea since drug delivery to the brain is always plagued by low bioavailability caused by the presence of the blood-brain-barrier (BBB). Because only small or lipophilic agents can cross the BBB, potent biomacromolecules such as nerve growth factors cannot be effectively delivered by conventional means. In response to dopamine deficiency associated with Parkinson's disease, local drug delivery systems such as pumps or controlled release polymers have been implanted intracranially to remedy the deficit. [Becker, J., et al., Sustained behavioral recovery from unilateral nigrostriatal damage produced by the controlled release of dopamine from a silicone polymer pellet placed into the denervated striatum. Brain Res., 1990. 508: p. 60.] While improved function has been reported in various experimental animal models, there are problems of dopamine autoxidation and cavitation around the injection site. As frequent replacements of devices in the brain is highly impractical, the limited service life-time of controlled release systems also renders this approach less attractive.
Transplantation of dopaminergic tissues into the striatum represents a potential solution. Nevertheless, although the transplanted fetal neurons can survive and make synaptic contacts with the host striatal neurons, there are formidable hurdles to be overcome. They include failure to reestablish the normal neural circuitry, high mortality and morbidity associated with the transplant procedure, and the ethical issue of human fetal tissue research. [Aebischer, P., et al., Transplantation of polymer encapsulated neurotransmitter secreting cells: Effect of the encapsulating technique. Transactions of the ASME, 1991. 113: p. 178.] It is also believed that over time the transplanted tissue will be rejected even if allogeneic tissue is used. To circumvent some of these obstacles, bovine adrenal medullary chromaffin cells and PC 12 cells were encapsulated in alginate/polylysine microcapsules or poly(acrylonitrile vinyl chloride) hollow fibers. [Aebischer, P., et al., Macroencapsulation of dopamine-secreting cells by co extrusion with an organic polymer solution. Biomaterials, 1991.12: p. 50; Tresco, P. A., S. R. Winn, and P. Aebischer, Polymer encapsulated neurotransmitter secreting cells. ASAIO J, 1992.38: p. 17.] In vitro studies show that at least some of the cells survived the encapsulation procedure. Release of dopamine from both the microcapsules and macrocapsules was observed in response to a chemically-induced depolarization. Encapsulated PC12 cells also alleviated lesion-induced rotational asymmetry in rats for least four weeks. [Aebischer, P., et al., Transplantation of microencapsulated bovine chromaffin cells reduces lesion-induced rotational asymmetry in rats. Brain Res., 1991. 560(1-2): p. 43.] Immunoprotection was demonstrated when both types of microcapsules were implanted in an immunologically incompatible host. [Dahiyat, B., et al., Synthesis and characterization of putrescine based poly(phosphoesterurethanes). J. Biomat. Sci., 1993: p. in press; Tresco, P. A., S. R. Winn, and P. Aebischer, Polymer encapsulated neurotransmitter secreting cells. ASAIO J, 1992.38: p. 17.]
Pain management is one of the major challenges of rehabilitation medicine. Conventional pharmacological intervention always require escalating doses and repeat administration. A recently reported promising approach for chronic pain management was to transplant adrenal medullary chromaffin cells into the spinal subarachnoid space. [Sagen, J., Chromaffin cell transplants for alleviation of chronic pain. ASAIO J, 1992.38: p. 24.] In rats the transplanted cells survived for months and released high levels of opioid peptides and catecholamines. In behavioral studies in rats, the transplants reduced pain in an arthritis model and a peripheral neuropathy model. Subsequent limited clinical trials demonstrated that the patients received pain relief over a period of 4-10 months, and a concomitant decrease in narcotic intake. Increased levels of catecholamines and metencephalon in the spinal CSF samples of patients also were observed. Success of this clinical trial was relied on the availability of human adrenal glands and the administration of the immunosuppressive agent cyclosporine A for two weeks. It appears that microencapsulated cells can bolster immensely the appeals of this cell-based management of chronic, and perhaps intractable, pain.
Human gene therapy depends on insertion of a desired gene into autologous cells. The success rate is low because of the difficulty of transfecting primary human cells. An alternative strategy is to genetically engineer easily transfectable cell lines from a non-autologous source to secrete a desired gene product. This approach was demonstrated in the secretion of significant levels of human growth hormone for weeks from mouse fibroblasts implanted in rat thymus. [Behara, A. M., A. J. Westcott, and P. L. Chang, Intrathymic implants of genetically modified fibroblasts. FASEB J., 1992.6: p. 2853; Doering, L. C. and P. L. Chang, Expression of a novel gene product by transplants of genetically modified primary fibroblasts in the central nervous system. I. NeuroSci. Res., 1991. 29(3): p. 292.] However, the novel gene product provoked an intense antibody response from its host recipient. Enclosing allogeneic recombinant cells in microcapsules should become an exciting approach in delivery of novel gene products.
Cells can be encapsulated in hollow fibers or in microcapsules that are several hundred microns in size. The former has the advantage of higher mechanical stability and retrievability. Microcapsules on other hand have a higher surface to volume ratio for growth of anchorage-dependent cells and lower mass transfer resistance for nutrients supply and product secretion. To combine the strength of the two approaches, microencapsulated cells can further be macroencapsulated, for instance, in hollow fibers; choice of highly permeable hollow fibers would add little to the overall mass transfer resistance. In the case of artificial pancreas design, this has the added advantage of preventing the islets from losing their bioactivity caused by aggregation. [Lacy, P. E., et al., Maintenance of normoglycemia in diabetic mice by subcutaneous xenografts of encapsulated islets. Science, 1991. 254: p. 1782.]
Microcapsule formulation is a known technology used by the pharmaceutical industry to manufacture sustained release products. However, the necessity of avoiding any harsh conditions that might damage cell viability eliminates many available methods. The most commonly used techniques for cell encapsulation are complex coacervation and interfacial precipitation. Complex coacervation involves the electrostatic interaction of two oppositely charged polyelectrolytes. At the right matching charge density, the two polyions combine and migrate to form a colloid-rich or water-insoluble phase. The molecular weight and chain conformation parameters of the polyions may also play an important role in the complexation process. Interfacial precipitation relies simply on the solidification of a dissolved polymer upon contact with an aqueous phase.
In the area of cell encapsulation, gelation of alginates is the most extensively studied system. Alginate is a glycuranan extracted from brown seaweed algae. Calcium or other multivalent counterions chelates contiguous blocks of alpha-1,4-L-guluronan residues present in the polysaccharide. Cell encapsulation is achieved when alginate solution containing suspended living cells is dropped or extruded into a solution containing calcium ions. The microcapsules formed can further be coated by adsorption of polyions such as polylysine, which can be coated by alginate again. Many cell types, including islets, hepatocytes, PC I12 cells, chondrocytes, and fibroblasts, have been encapsulated by this method.
The standard single step drop technique has been rejected not to be reproducible [Wong, H. and T. M. S. Chang, Microencapsulation of cells within alginate poly-l-lysine microcapsules prepared with standard single step drop technique histologically identified membrane imperfections and the associated graft rejections. J. Biomar. Artificial Cells Immob. Biotech., 1991. 19: p. 675.] A variable number of cells become embedded in the membrane matrix and some are exposed to the surface of the microcapsules.
When implanted in mice, this led to an undesirable acute cell-mediated host response. Macrophages and lymphocytes were also observed to perforate the membrane and infiltrate the microcapsules. A two-step microencapsulation procedure was reported to circumvent this problem. In essence, the above cell-containing microsphere is encapsulated one more time to form larger calcium alginate gel microspheres. After the larger microspheres are coated by poly-1-lysine outside, the contents inside are liquified by titrate to remove calcium, resulting in free floating cells that are not embedded in the capsule membrane. [Wong, H. and T. M. S. Chang, A novel two-step procedure for immobilizing living cells in microcapsules for improving xenograft survival. J. Biomat. Artificial Cells Immob. Biotech., 1991. 19: p. 687.]
Macrocapsules composed of totally synthetic polyelectrolytes were prepared by the complex coacervation principle. Polymers containing methacrylic acid (MAA) and dimethylaminoethyl methacrylate (DMAEMA) functionality show promise as microcapsule-forming pairs for the entrapment of mammalian cells. For example, such materials have been used to encapsulate erythrocytes. [Shao, W., Y. Xiaonan, and W. T. K. Stevenson, Microcapsules through polymer complexation I: Complex coacervation of polymers containing a high charge density. Biomaterials, 1991. 12: p. 374; Shao, W., et al., Microcapsules through polymer complexation II: By complex coacervation of polymers containing a low charge density. Biomaterials, 1991.12: p. 479.]
Early in vivo results with the alginate-polylysine system have not always been consistent because of the uncontrolled purity of alginate, and presumably also because of the incorporation of cells into the external membrane. The in vivo mechanical stability of microcapsules made by the new two-step technique remains to be tested, because the calcium ions are stripped. Even for microcapsules made by the standard technique, there is evidence that materials resembling alginate were around the microcapsules in the brain parenchyma of rats four weeks post-implantation. [Aebischer, P., et al., Transplantation of polymer encapsulated neurotransmitter secreting cells: Effect of the encapsulating technique. Transactions of the ASME, 1991. 113: p. 178.] Totally synthetic membranes can be more stable but they might not be the optimal substrates for cell growth and function.
Interfacial precipitation also has been used to form microencapsulates. In this method, cell suspension and polymer solution are extruded separately through two concentrically configured needles into a precipitating bath. Organic solvents such as dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), dimethyl acetamide (DMAc), diethyl phthalate, and acetone are used to dissolve the organic polymers. Contact of cells with organic solvents is unavoidable but can be minimized through various coextrusion schemes.
Encapsulation of chromaffin and PC12 cells was achieved by this technique using poly(acrylonitrile-co vinyl chloride) as the membrane, in configurations of 1 cm long hollow fibers. [Aebisher, P., et al, Transplantation of polymer encapsulated neurotransmitter secreting cells: Effect of the encapsulating technique. Transactions of the ASME, 1991. 113: p. 178; Aebischer, P., et al., Macroencapsulation of dopamine-secreting cells by coextrusion with an organic polymer solution. Biomaterials, 1991. 12: p. 50.] In vitro and in vivo studies show that both cell viability and cell functions were largely preserved in the encapsulation process despite the fact that the cells were in contact with DMF or DMAc. No microcapsules have been prepared from this polymer-solvent system.
RL.RTM., a water-insoluble polyacrylate available from Monsanto, has been used to form membrane for encapsulating erythrocytes and fibroblasts. [Boag, A. H. and M. V. Sefton, Microencapsulation of human fibroblasts in a water-insoluble polyacrylate. Biotech. Bioeng, 1987. 30: p. 854.] Diethyl phthalate was used as the organic solvent and a mixture of corn oil and mineral oil was used as the precipitating bath. Fibroblasts did not grow in the microcapsules unless collagen was also co-encapsulated and the microcapsules are fragile. [Broughton, R. L. and M. V. Sefton, Effect of capsule permeability on growth of CHO cells in Eudragit RL microcapsules: use of FITC-dextran as a marker of capsule quality. Biomaterial, 1989. 10: p. 462]
Subsequently, cationic polyacrylates involving the copolymers of MMA-DMAEMA were used to improve mechanical stability. [Mallabone, C. L., C. A. Crooks, and M. V. Sefton, Microencapsulation of human diploid fibroblasts in cationic polyacrylates. Biomaterials, 1989. 10: p. 380.] Growth of the encapsulated human diploid fibroblasts and Chinese hamster ovary (CHO) cells, however, was deemed unsatisfactory due to poor permeability for nutrients. [Broughton, R. L. and M. V. Sefton, Effect of capsule permeability on growth of CHO cells in Eudragit RL microcapsules: use of FITC-dextran as a marker of capsule quality. Biomaterials, 1989. 10: p. 462.]
Continued improvement led to macroporous MMA-hydroxyethyl methacrylate (MMA-HEMA) microcapsules that have higher permeability. [Crooks, C. A., et al., Microencapsulation of mammalian cells in a HEMA-MMA copolymer: Effects on capsule morphology and permeability. J. Biomed. Matr. Res., 1990. 24: p. 1241.] Evaluation of encapsulated hepatoma cells (HepG2) indicated that the cells formed aggregates instead of adhering to the copolymer, with central necrosis at day 7 in in vitro culture. [Babensee, J. E., U. D. Boni, and M. V. Sefton, Morphological assessment of hepatoma cells (HepG2) microencapsulated in a HEMA-MMA copolymer with and without Matrigel. J. Biomed. Matr. Res. 1992. 26: p. 1401.] Co-encapsulating Matrigel, a reconstituted extracellular matrix derived from mouse tumor basement membrane, did improve the cell viability.
A two-step process to encapsulate cells involving in situ polymerization has been reported. [Caidic, C., C. Baquey, and B. Dupuy, "Inverted Microcarriers" for cell cultures made by polymerization of shells around agarose microspheres in a non-cytotoxic procedure. Polymer, 1991. 23(8): p. 933] First the cells were encapsulated by extruding cell suspensions in agarose solution into 4.degree. C. paraffin oil. A polymer shell composed of polyacrylamide was formed around each bead using a latex-seeded photoinduced polymerization process. Cell viability was demonstrated by continuous secretion of prolactin from encapsulated rat pituitary cells for three days; the cells were not yet responsive to alterations in potassium levels at this time point.
Whereas synthetic microcapsules are mechanically and chemically more stable than the polyelectrolyte gels composed of polysaccharides, low permeability is consistently an issue. These synthetic polymers are also not optimal substrates for cell attachment, growth and functions.
There remains a need for effective microencapsulation of living cells.